Hyperthermia is known to sensitize mammalian cells to radiation damage. However, the fractionation of combined hyperthermia and radiation with a 24 hr fractionation interval and normal culture conditions eliminates the heat-induced sensitization to radiation. The overall objective of this proposal is to determine the in vitro cellular response to fractionation of combined hyperthermia and radiation under tumor-like culture conditions (low pH, nutrient deprivation). This cellular response is expected to reflect the sensitization to heat-induced damage by low pH and nutrient deprivation, the desensitization to radiation-damage (repair of PLD and oxygen effect), and the possible modification of the fractionation response observed under normal culture conditions. We hypothesize that the net effect of fractionated heat and radiation is to sensitize cells differentially under tumor-like vs. normal culture conditions. A confirmation of this hypothesis would offer a rationale for the observation that tumor cells in vivo appear more sensitive than normal tissues to the fractionation of combined hyperthermia and radiation. In addition, an understanding of the interaction of environmental (culture) conditions with the fractionation response to heat and radiation will provide a basis for the design of specific fractionation protocols which are differentially cytotoxic under tumor-like vs. normal culture conditions. A subordinate hypothesis to be tested is that the cellular survival response can be predicted by a specific radiation or heat-induced molecular lesion independent of the specific culture and fractionation conditions. A confirmation of this hypothesis with the identification of a prognostic biochemical assay for cell survival could provide the foundation for a subsequent in vivo study of fractionation effects by combined hyperthermia and radiation in normal vs. malignant tissues.